May 31, 2025
Manual Wastewater Concentration and Extraction V1.0
- Allison Steedman1,
- Mark Zeller1
- 1Scripps Research
Protocol Citation: Allison Steedman, Mark Zeller 2025. Manual Wastewater Concentration and Extraction V1.0. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldndjxv5b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 31, 2025
Last Modified: May 31, 2025
Protocol Integer ID: 219235
Keywords: wastewater, extraction, ceres, concentration, magmax, rna, dna, manual, magnetic-bead based, dna from collected wastewater, collected wastewater, manual wastewater concentration, wastewater, throughput version of this protocol, rna, extraction, throughput version, protocol, dna
Funders Acknowledgements:
Bill & Melinda Gates Foundation
Grant ID: INV-057213
Abstract
This protocol is designed to manually extract RNA/DNA from collected wastewater.
For a high-throughput version of this protocol, please refer to this protocol: https://www.protocols.io/view/high-throughput-wastewater-sars-cov-2-detection-pi-81wgb74xyvpk/v1
Materials
Wastewater, Ceres Nanotrap Beads: SKU#44202-30, MagMax Lysis Buffer: A42361, MagMAX Viral/Pathogen Kit: A42352
Troubleshooting
Concentration
Invert wastewater sample to mix
Aliquot 10mL of wastewater into a 15mL conical tube
Add 150ul Ceres Nanotrap Microbiome A Particle beads into 10mLs of wastewater and invert to mix
Incubate at room temp for 15 minutes on slow shaker (or invert every 5 minutes)
Place 15mL conical on magnetic stand for 5 minutes or until beads are fully separated
Pipette off the supernatant without disturbing the bead pellet
Remove from magnet and add 500ul MagMax Lysis Buffer. Resuspend the pellet and transfer to a 1.5mL tube.
Incubate at room temp for 10 minutes on a slow shaker
Place 1.5mL tube on magnetic stand for 2 minutes or until beads are fully separated
Collect supernatant into new 1.5mL tube and discard bead pellet. Continue right to extraction.
Extraction
Prepare the following mixture in a 1.5mL conical tube:
| Reagent | Volume (ul) | |
| Binding Solution | 500 | |
| Beads | 20 | |
| Proteinase K | 10 | |
| Sample/Lysate | 400 |
*Reagents (aside from sample) from MagMAX Viral/Pathogen Kit (A42352)
Vortex to mix
Incubate for 10 minutes
Place 1.5mL conical on magnetic rack for 2 minutes or until beads are fully separated
Discard supernatant
Take off magnetic rack and add 1000ul MagMax Wash Buffer and resuspend bead pellet
Place 1.5mL conical on magnetic rack for 2 minutes or until beads are fully separated
Discard supernatant
With the tube still on the magnet, add 1000 μl of 80% EtOH, incubate for 30 seconds, and remove the EtOH wash.
Repeat previous step for another 80% EtOH wash step
Leave the plate on the magnet to air dry for 3 min.
Remove the tube from the magnet. Add 50ul of MagMax Elution solution and pipette and vortex to resuspend
Incubate at room temperature for 10 minutes
Place the tube onto the magnet. Transfer the supernatant to the final tube. The sample is ready for downstream analysis or can be stored at -80⁰ C